Transport of the Glucosamine-Derived Browning Product Fructosazine

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Transport of the glucosamine-derived browning product fructosazine (polyhydroxyalkylpyrazine) across the human intestinal Caco-2 cell monolayer: role of the hexose transporters Abhishek Bhattacherjee, Yuliya Hrynets, and Mirko Betti J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 23 May 2017 Downloaded from http://pubs.acs.org on May 30, 2017

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Journal of Agricultural and Food Chemistry

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Transport of the Glucosamine-derived Browning Product Fructosazine

2

(Polyhydroxyalkylpyrazine) Across the Human Intestinal Caco-2 cell

3

Monolayer: Role of the Hexose Transporters

4

Abhishek Bhattacherjeea, Yuliya Hrynetsa and Mirko Bettia*

5 6

7

Affiliations

8

a

9

410 Agriculture/Forestry Centre

10

Edmonton, AB T6G 2P5 Canada

Department of Agricultural, Food and Nutritional Science, University of Alberta

11

12

*Corresponding Author

13

Dr. M. Betti

14

E-mail: [email protected]

15

Tel: (780) 248-1598

16

Fax: (780) 492-4265

17 18 19

Short Title: Involvement of hexose transporters in fructosazine transport

20 21 22

1 ACS Paragon Plus Environment


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Abstract

24

The transport mechanism of fructosazine, a glucosamine self-condensation product, was

25

investigated using a Caco-2 cell model. Fructosazine transport was assessed by measuring the

26

bidirectional permeability coefficient across Caco-2 cells. The mechanism of transport was

27

evaluated using phlorizin, an inhibitor of sodium-dependent glucose cotransporters (SGLT) 1

28

and 2, phloretin and quercetin, an inhibitors of glucose transporters (GLUT) 1 and 2, transcytosis

29

inhibitor wortmannin, and gap junction disruptor cytochalasin D. The role of hexose transporters

30

was further studied using downregulated or overexpressed cell lines. The apparent permeability

31

(Pa-b) of fructosazine was 1.30 ± 0.02 × 10-6 cm/s. No significant (p > 0.05) effect was observed

32

in fructosazine transport by adding wortmannin and cytochalasin D. The presence of phlorizin,

33

phloretin and quercetin decreased fructosazine transport. The downregulated GLUT cells line

34

was unable to transport fructosazine. In human intestinal epithelial Caco-2 cells, GLUT1 or

35

GLUT2 and SGLT are mainly responsible for fructosazine transport.

36 37 38

Keywords: Glucosamine, Fructosazine, Caco-2 cell model, Permeability, GLUT, SGLT

39 40 41 42 43 44 45


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Introduction

47

Pyrazines (1,4-diazines) are a class of heterocyclic compounds typically found in toasted

48

and roasted foods as a consequence of nonenzymatic browning reactions; these compounds

49

significantly contribute to the flavor and odor of food. Flavorful pyrazines are also found in

50

certain plants and animals, and in foods processed by fermentation.1 When food is heated the α-

51

aminocarbonyls are central compounds in the formation of these pyrazines. These 2-amino

52

ketones can be produced through the Strecker degradation pathway involving the initial reaction

53

of reducing sugars or α-dicarbonyl with free amino acids, or through the mechanism proposed by

54

Van Lancker et al.2 and Scalone et al.3 involving the reaction between α-dicarbonyl and peptides.

55

Alpha-aminocarbonyl compounds undergo a condensation reaction leading to the formation of

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dihydropyrazine and depending on the condition, can then form a pyrazine molecule through: I)

57

an oxidative reaction (Figure 1A path I) or II) an aldol-type reaction with the Strecker aldehyde

58

if a dihydropyrazine anion is formed (Figure 1A path II). The reaction between reducing sugars

59

(for instance fructose with ammonia) also generates α-aminocarbonyl compound glucosamine

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(1-amino-1-deoxy-D-glucose),

61

polyhydroxyalkylpyrazines,

62

tetrahydroxybutyl)pyrazine)) and deoxyfructosazine (2-(D-arabino-tetrahydroxybutyl)-5-(D-

63

erythro-2,3,4-trihydroxybutyl))pyrazine (Figure 1B).4,5

which such

can

then

also

fructosazine

self-condense

forming

the

(2,5-bis-(D-arabino-1,2,3,4-

64

In general, a variety of substituted pyrazines are produced during the heating of food and

65

depending on the type of substitution, pyrazines can be volatile or non-volatile. Examples of

66

volatile pyrazines are 2-methylpyrazine found in “flavor-faded” roasted peanuts6 or 2,3- and 2,6-

67

dimehtylpyrazines identified in cooked beef.7 On the other hand, fructosazine [1] is a non-

68

volatile pyrazine found in caramel,8 soy sauce9 and peanuts.10 Recently, Hrynets et al.5 produced



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and quantified 1 from the non-enzymatic browning reaction of chitin-derived glucosamine. Van

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Der Ark et al.11 patented the use of glucosamine-derived fructosazines, specifically 2,5- and 2,6-

71

deoxyfructosazines, as additives in beverages (i.e. beer) and foodstuffs to heighten the resistance

72

to light-induced flavor changes. Bhatacherjee et al.12 discovered that 1 possesses antimicrobial

73

activity against an extremely heat resistant E. coli AW 1.7. In addition to their application as

74

food ingredients, compound 1 and deoxyfructosazine are gaining interest for their uses in human

75

therapeutics such as in the treatment of type II diabetes and the prevention of atherosclerosis.13

76

Furthermore,

Giordani

et

al.14

and

Zhu

et

al.15

showed

that

these

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(polyhydroxyalkyl)pyrazines possess an inhibitory activity against interleukins (1β and 2), which

78

in turn could be helpful to prevent pathological cartilage degradation and other

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inflammatory diseases. Thus glucosamine-derived pyrazines may be more effective than

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glucosamine itself in treatment of osteoarthritis. Indeed, several studies have indicated that

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deacetylated glucosamine can alleviate the symptoms of osteoarthritis. However the ability of

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glucosamine to produce these heterocyclic compounds even at moderate temperature poses a

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dilemma on which between glucosamine or products of its self-condensation are responsible for

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the therapeutic effect.

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In light of these new properties, new environmentally-sustainable chemical processes

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have been proposed to synthesize these heterocyclic compounds. For instance, both 1 and

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deoxyfructosazine can be economically converted from glucosamine in the presence of

88

boronate/phenylboronate,16 basic ionic liquids such as 1-butyl-3-methylimidazolium hydroxide17

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or 1-ethyl-3-methylimidazolium acetate18 and imidazolium ionic liquid.19 The advantage of using

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such processes lies in the limited amount of the browning compounds (i.e. melanoidins)


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produced during the nonenzymatic browning of glucosamine, thus increasing the purity and yield

92

of production of 1 and deoxyfructosazine.18

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Despite the growing interest in polyhydroxyalkylpyrazine research, no studies have been

94

conducted so far to elucidate the mechanism of its transepithelial transport. In general, for any

95

ingested bioactive molecule to render its effects on the body it must be able to transport across

96

the intestinal barrier and then appear in the circulation.20 Yet few studies have been conducted so

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far to understand the mechanism of transport of nonenzymatic browning products, and most of

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them have been devoted to glycated amino acids and peptides.21-24 A closer look at the research

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conducted on N-heterocyclic compounds originated from the Maillard reaction reveals that 2-

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amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (Figure S1A) is a carcinogenic product

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produced from the reaction between phenylalanine, creatinine and carbohydrates, and is fairly

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well absorbed in Caco-2 intestinal epithelial monolayer.25 Moreover, Chen et al.26 showed that 5-

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(5,6-dihydro-4H-pyridin-3-ylidenemethyl)furan-2-yl]-methanol (also known as F3-A) (Figure

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S1B) isolated from breads can efficiently pass across the Caco-2 cell monolayers. As previously

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mentioned, studies on the transport of 1,4-diazines are very scarce, and completely absent are

106

studies on the transport mechanism for 1. Therefore, the objective of this study was to

107

understand the transport of 1 across the epithelial barrier in the Caco-2 intestinal monolayer.

108

Our hypothesis is that compound 1 is transported through an active process that involves the

109

hexose transporters such as glucose transporters (GLUT) and sodium-dependent hexose

110

transporters (SGLT). The rationale behind this hypothesis is based on the peculiar structure of 1,

111

which possesses both hydrophilic –OH and hydrophobic (pyrazine ring) residues capable of

112

interacting with the hexose transporters without obstructing them, in a similar manner as glucose

113

does.27-29 Hence, it is possible that 1 has a similar mechanism. For this purpose the role of



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different hexose transporters GLUT and SGLT1, in the transport mechanism of 1 was studied in

115

human intestinal cell lines.

116

Chemicals

117

Fructosazine [1] (> 98%) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA,

118

USA). Caco-2 human colon-derived adenocarcinoma cells (HTB37) were purchased from

119

American Type Culture Collection (Manassas, VA, USA). Dulbecco’s Modified Eagle Medium

120

(DMEM), DMEM without glucose, 0.25% (w/v) trypsin−0.53 mM ethylenediaminetetraacetic

121

acid

122

piperazineethanesulfonic acid (HEPES), fetal bovine serum (FBS), and 1% Pen-Strep antibiotics

123

(10000 units/mL of penicillin, 10000 of µg/mL strepromycin) were from Gibco Invitrogen

124

(Burlington, ON, Canada). Glucose estimation kit, wortmannin, cytochalasin D, phloretin,

125

phlorizin dihydrate, Tween 20, Triton X-100 were from Sigma-Aldrich (St. Louis, MO, USA).

126

MTT cell proliferation assay kit was purchased from ATCC Bioproducts (Manassas, VA, USA).

127

Primary antibodies against GLUT1-5, SGLT1 transporters and β-actin (ab14683, ab192599,

128

ab191071, ab188317, ab14686 and ab36057) were from Abcam (Boston, MA, USA). Anti-

129

Rabbit IgG H&L (DyLight 488) preadsorbed fluorescent secondary antibody (ab202372),

130

Ponceau S (ab146313) and 10× RIPA lysis buffer (ab 156034) were also purchased from Abcam.

131

Mini-Protean TGX precast gels for western blotting analysis were from Bio-Rad (Richmond,

132

CA, USA). iBlot gel transfer stacks (PVDF, regular), Lucifer yellow CH lithium salt and other

133

reagents were from Fisher Scientific (Ottawa, ON, Canada). Transwell polyester permeable

134

membrane supports (0.4 µm pore size and 12 mm diameter) were from Corning (NY, USA).

135

Uniformly [14C]-labelled glucose (250 mCi/mmol) was purchased from American Radiolabelled

136

Chemicals Inc. (St. Louis, MO, USA).

(EDTA),

Hank’s

Balanced

Salt

Solution

(HBSS),

4-(2

hydroxyethyl)-1-


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Materials and Methods

138

Cell Cultures

139

The Caco-2 cells were cultured in DMEM with HEPES (25 mM) supplemented with 10% FBS

140

and 1% Pen Strep antibiotics (10000 units of penicillin/mL, 10000 µg/mL of streptomycin). All

141

biosafety studies were carried out in a Nuaire Biosafety Cabinet Class II type A (Plymouth, MN,

142

USA). The cell lines were maintained at 37°C in a Forma Water-Jacketed incubator (Thermo

143

Fisher Scientific Inc., Waltham, MA, USA) under 5% CO2, and 90% relative humidity. Cells

144

were grown up to 80% confluence and trypsinization with trypsin-EDTA treatment was used

145

during subculturing to remove the adherent cells. For the transport experiments, the Caco-2 cells

146

were seeded at a density of 1 × 105 cells/insert (1.12 cm2) onto a 12-well Transwell polyester

147

permeable membrane support. Cell culture medium was replaced at two day intervals and cells

148

were allowed to differentiate for at least 21 days. All experiments were performed at cell

149

passages 18-21. The volumes amounted at the apical side and basolateral side were 0.5 and 1.5

150

mL, respectively.

151

Quality Control of Caco-2 Cell Monolayers

152

The heterogeneity of the cells was determined using a light microscope (Zeiss PrimoVert,

153

Oberkochen, Germany) to ensure typical smooth dense monolayers. The integrity of Caco-2 cell

154

monolayers was evaluated by measuring the transepithelial electrical resistance (TEER) with a

155

Multi-channel Voltage/ Current ECV-4000 system (World Precision Instruments, FL, USA) and

156

calculated as:

157

TEER (Ω·cm2) = [TEER Ω – TEER Ωno cells] × area (cm2)

158

where TEER is the electrical resistance across Caco-2 monolayers and TEERno

159

electrical resistance across the insert without cells. The area refers to the area of the insert.30 Cell

(1) cells

is the



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monolayers with TEER of more than 300 Ω·cm2 were considered confluent and were used for

161

the transport studies. Along with TEER values, the integrity of Caco-2 cell monolayers was

162

monitored using a hydrophilic Lucifer yellow (300 µM), a paracellular permeation marker. The

163

permeation of this fluorescent dye was measured in the basolateral compartment at

164

excitation/emission wavelength of 430/540 using a fluorescence microplate reader Spectra Max

165

M3.30

166

The MTT Cell Proliferation Assay

167

Cytotoxicity of 1 was tested on Caco-2 cells by the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl

168

tetrazolium bromide (MTT) assay.31,32 Caco-2 cells (passage 20) were grown in 96-well culture

169

plates and incubated with different concentration of 1 (0.5-10 mg/mL) at 37°C for 12 h and the

170

cell viability was monitored using commercial MTT cell proliferation assay kit. The kit test was

171

used to determine the ability of viable cells to reduce the yellow tetrazolium salt (MTT) to blue-

172

colored formazan crystals by mitochondrial enzymes. The concentration of formazan crystals

173

was then spectrophotometrically determined when dissolved in an organic solvent. Different

174

concentrations of 1 were added to the wells and incubated for 12 h at 37˚C in a humidified

175

atmosphere under 5% CO2. After the incubation period, 10 µL of MTT reagent was added to

176

each well and incubated at 37˚C for 4 h. Once the purple crystals were visualized they were

177

dissolved in 100 µL DMSO solution. Samples were kept in dark at a room temperature for 120

178

min. After incubation, the absorbance was measured at 570 nm by using Spectra Max M3

179

spectrophotometer. The buffer blank was the negative control and was subtracted to remove

180

buffer interference, while the cells without 1 treatment were considered as the positive control

181

cells. Cell viability was calculated from a linear regression equation (y = mx + b) from the dose-

182

response curve plotted using the concentrations of 1 versus absorbance values.


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Transport Experiments and Molecular Mechanism

184

The transport experiments were performed on Caco-2 cells (prepared as reported in the earlier

185

section) according to previous studies.20,32,33 Bidirectional transport of 1 was monitored across

186

Caco-2 cell monolayers. Briefly DMEM medium was removed prior to transportation studies

187

and cells were equilibrated for 30 min in HBSS medium. The transport of 2 mg/mL of 1 was

188

followed as a function of time where samples were collected from the apical/basolateral side

189

every 30 min during 120 min. The apparent permeability coefficient (Papp, cm/s) was determined

190

from the amount of 1 transported from apical to basolateral (influx) or basolateral to apical

191

(efflux) directions according to the following equation:

192

app =

193

where dQ/dt is a steady–state flux (ng/s), A is the surface area of the insert (cm2) and C0 is the

194

initial concentration in the donor compartment (ng/cm3).34

195

The molecular mechanism of 1 transport was evaluated in another experimental setup using

196

different inhibitors. Mode of action of all these inhibitors is reported and validated by several

197

research groups in different in vitro and in vivo conditions. Briefly, cells were pre-incubated with

198

gap junction disruptor cytochalasin D (0.25 µg/mL in DMSO),35 transcytosis inhibitor

199

wortmannin (250 nM in DMSO),20 SGLT1 and 2 blocker phlorizin (0.5 mM in DMSO), GLUT

200

inhibitor phloretin (1 mM in DMSO)36 and quercetin (0.2 mM in DMSO)37 for 30 min, and then

201

transport studies were performed by addition of 2 mg/mL solution of 1. All of the inhibitors were

202

dissolved in DMSO and diluted with transport buffer before adding to the Caco-2 cell monolayer

203

(final concentration of DMSO was 0.044%). Samples were collected after 120 min from each

204

experimental set and subjected to analyses by UHPLC. Background interference was removed by

(2)



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205

subtracting the value of the blank which was prepared under identical condition as described

206

before except addition of 1.

207

Under conditions described in the section above, for comparison purposes the transport of

208

radioactive [14C]glucose was also monitored in presence and absence of cytochalasin D,

209

wortamanin, phlorizin, phloretin and quercetin by a radio-chemical detection in liquid

210

scintillation counter (Beckman 6500, USA).

211

Along with the efficiency, the toxicological effect of these inhibitors on Caco-2 cell was also

212

evaluated by MTT assay. Briefly, Caco-2 cell (cell passages 20) was treated with these inhibitors

213

(identical concentration) and cell viability was monitored by MTT assay as reported in the earlier

214

section.

215

Analysis by UHPLC

216

The analysis of the samples collected from transport and molecular mechanism experiments were

217

performed according to previous studies.5,12 The UHPLC system (Shimadzu, Columbia, MD,

218

USA) used consisted of a binary pump (LC-30AD), autosampler (SIL-30AC), a photodiode array

219

detector (SPD-M20A) and a column compartment (CTO-20AC). Ten microliters of the injected

220

samples were separated on an Ascentis Express ES-C18 column (150 × 4.6 mm, 2.7 µm

221

particles) (Sigma-Aldrich, MO, USA) at 25.0 ± 0.5°C, a flow rate of 0.7 mL/min and a detection

222

wavelength of 275 nm.5,12 A binary gradient consisted of solvent A (0.1% formic acid in water,

223

v/v) and B (100% methanol). For elution a gradient of 0-5% B from 5 to 15 min, 5-50% B from

224

15 to 25 min and 50-5% B from 25 to 35 min was used. The standard concentrations of 1 were

225

analyzed by UHPLC, and peak area measurements were used to build a calibration curve for

226

samples quantitation. The calibration curve was linear with a correlation coefficient of 0.996.


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The limits of quantitation (LODs) and limits of detection (LOQs) were 0.82 ± 0.02 and 2.71 ±

228

0.07 µg/mL, respectively. The LODs and LOQs were defined as the concentrations injected that

229

provided signals equivalent to 3 and 10 times of the baseline noise (signal-to-noise ratios of 3

230

and 10), respectively.

231

HPLC-ESI-MS/MS

232

To ensure correct peak identity mass spectrometry analyses were conducted using HPLC unit

233

(Agilent, Palo Alto, CA, USA) connected to a 4000 Q TRAP LC-MS/MS System (Applied

234

Biosystems, Concord, ON, Canada). The HPLC separation was performed under the same

235

separation conditions as described above for UHPLC. Ionization was achieved using ESI in the

236

positive mode at a spray voltage of 4 kV and source temperature 500°C. Full scan data were

237

acquired by scanning from m/z 50 to 500. The selected precursor ions were fragmented to

238

produce products ions by collision-induced dissociation (CID) using nitrogen as a collision gas

239

under collision energy of 30 eV.

240

Caco-2 cell Culture for Differential Expression of GLUT Transporters

241

To achieve differential expression of GLUT transporters in the Caco-2 cell system, culture

242

conditions were modified. To completely downregulate the expression of GLUT transporters in

243

Caco-2 cells the method of Mesonero et al.38 was used. Briefly, Caco-2 cells were cultured in a

244

standard condition for 10 days following the methodology described in the earlier section. This

245

time period (10 days) allowed the cells to start their differentiation process. After 10 days of

246

standard culturing, 10% dialyzed FBS serum and hexose free DMEM medium was introduced to

247

the culture condition. Cells were grown in these modified conditions for 10 days. Hexose free

248

medium significantly inhibits the GLUT transporter formation in Caco-2 cell line. Expression of

249

GLUT (1-5) and SGLT1 were thoroughly tested in this cell line by western blot technique and it



Page 12 of 36

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was further used during transport studies to represent the cells (designated as –GLUT cell line)

251

with downregulated expression of GLUT transporter. For the transport experiments, cells were

252

seeded at a density of 1 × 105 cells/insert (1.12 cm2) onto a 12-well Transwell polyester

253

permeable membrane support. Cells were grown in glucose free DMEM medium for at least 21

254

days. The heterogeneity of the cells was determined using a light microscope to ensure typical

255

smooth dense monolayers. After 3 weeks of culture, the integrity of Caco-2 cell monolayers was

256

evaluated by measuring the TEER as described in earlier section.

257

Another modified Caco-2 cell line was also cultured according to Mesonero et al.38 which

258

represents overexpression of GLUT5 transporter. There was no suitable inhibitor that specifically

259

inhibits GLUT5 transporter without affecting other GLUTs. To overcome this challenge GLUT5

260

overexpressed cell line was used to assess the role of GLUT5 transporter in the transport of 1.

261

Briefly, cells were cultured under standard conditions for 10 days to allow the time for

262

differentiation. After 10 days FBS serum (10% dialyzed) was introduced in the culture condition

263

and further glucose free medium was supplemented by 25 mM fructose, since addition of

264

fructose helps the overexpression of GLUT5.38 Cells were further cultured in the modified

265

condition until transportation studies, which were conducted as described in earlier sections.

266

Expression of GLUT (1-5) and SGLT1 were thoroughly tested in this cell line by western

267

blotting. This Caco-2 cell line was designated as ++GLUT5 cell line (overexpressed GLUT 5

268

transporter).

269

On the final day of experiment modified medium was removed and transportation studies were

270

performed identically as described in the earlier section. A control set was also cultured under

271

standard condition and represented the cell line with expression of GLUT (GLUT 1-5) and


Page 13 of 36


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SGLT1 transporters and designated as control cell line throughout the text. All experiments were

273

performed at cell passages 18-21.

274

Evaluation of Protein Expression of Transepithelial Transporters by Western Blotting

275

For cell lysis, the cultured cells were washed with phosphate-buffered saline (PBS) and

276

commercially obtained ice cold RIPA buffer (Abcam, Cambridge, MA, USA). Whole cell lysate

277

was transferred to a pre-cooled microfuge tubes and further agitated for 30 min at 4˚C. After

278

incubation time tubes were centrifuged (10000g at 4˚C for 20 min). The supernatant was

279

carefully collected for further analysis and the pellet was discarded. The protein content of the

280

sample was analysed by BCA protein estimation kit (Thermo Fisher Scientific) as per the

281

manufacturer’s protocol. An equal amount of protein (20 µg) was loaded to Bio-Rad precast mini

282

gels and resolved by Biorad-gel electrophoresis system. After SDS gel electrophoresis the gel

283

was transferred onto a polyvinylidene difluoride (PVDF) membrane by using iBlot Dry Blotting

284

System (Invitrogen, USA) and designated transfer kit (iBlot Transfer Stack, Invitrogen). After

285

the transfer membrane was blocked with 5% skimmed milk (Bio-Rad, Hercules, CA, USA) it

286

was placed in TBS-T (Tris-buffered saline containing Tween-20) for 2 h at 4˚C under constant

287

agitation. After incubation, the membrane was further rinsed with TBS-T buffer three times for 5

288

min each. The primary antibody was added to the membrane diluted 1000-fold with TBS-T and

289

the membrane was kept under constant agitation overnight (16 h). The primary antibody was

290

removed after the incubation period and the membrane was further washed with TBS-T for 15

291

min (3 × 5 min). The fluorescence tagged secondary antibody was used at 1:10000 dilution

292

(diluted is TBS-T) and the membrane was kept under constant agitation for 2 h at room

293

temperature. Finally, fluorescent bands were visualized by Typhoon FLA 9500 (GE Healthcare,

294

Life Sciences) and the recorded images were analyzed by Image Q software.



Page 14 of 36

295

Transepithelial Transport of Radioactive [14C]Glucose in Presence of Fructosazine

296

The growth conditions for Caco-2 cells and transport experiments were conducted in a similar

297

way as described in an earlier section. Briefly, the transport pattern of radioactive [14C]glucose

298

(3.4 µM) was monitored in the presence and absence of 1 (2 mg/mL). Experimental samples

299

were loaded in the apical chamber containing transport buffer (HBSS, pH 7.4) and Caco-2 cells

300

were further incubated at 37˚C for 2 h. Samples were collected from the basal compartment

301

every 30 min during the 120 min duration of the experiment. Collected samples (10 µl) were

302

further diluted 500 times with scintillation fluid (4.990 mL) to make a 5 mL reaction mixture.

303

Their cumulative transport amount was assessed by detecting the radioactivity of the resultant

304

reaction mixture in a liquid scintillation counter (Beckman LS6500, Fullerton, CA, USA) using

305

the method of Walgren et al.39 A negative blank without glucose or 1 was prepared and

306

considered as baseline. Counting efficiency of the radioactive molecule was 78%. The apparent

307

permeability coefficient (Papp

308

earlier section.

309

Statistical Analysis and Data Presentation

310

Values reported are the mean ± standard deviation (SD) of three independent experiments. All

311

the data were analyzed using one-way analysis of variance (ANOVA) by SAS 9.4 (Cary, NC).

312

Post hoc multiple comparison test was Tukey's test, where p ≤ 0.05 was considered significantly

313

different.

314

Result and Discussion

315

Cytotoxicity of Fructosazine in Caco-2 Cells

316

Prior to the experiments on the transepithelial transport of 1 in Caco-2 cells, the viability of cells

317

was measured using MTT assay to evaluate the cellular cytotoxicity in the concentration range

a-b)

of [14C]glucose was determined by the equation mentioned


Page 15 of 36


318

from 0.5 to 10 mg/mL. As shown in Figure 2 for the concentrations tested up to 2 mg/mL, cell

319

viability was greater than 97%, suggesting no toxic effect on the cell physiology, and thus could

320

be safely used in transport studies. In a recent study12 3.6 mg/mL of 1 was able to inhibit the

321

growth of extremely heat-resistant E.coli AW 1.7 by 50%. A similar trend was observed in MTT

322

assay with an increasing concentration of 1 to around 5.6 mg/mL. In the present study, 5.6 and

323

10 mg/mL of 1 resulted in 49.1 ± 1.0 and 33.7 ± 1.3% cell viability, respectively. Based on these

324

results to ensure cell viability, a nontoxic concentration of 2 mg/mL was chosen for further

325

experiments.

326

Transport of Fructosazine across Caco-2 Cell Monolayers

327

Quality control was performed with wells having TEER values >300 Ω·cm2 to ensure the

328

confluence of Caco-2 cells monolayers prior to transport studies. TEER values of the Caco-2 cell

329

monolayers before and after transport experiments were monitored and reported in the Table S1.

330

Another method was also used to ensure the integrity of the cell monolayer, where the

331

fluorescent of Lucifer Yellow dye was applied and showed a flux of < 2.1% before (0 min) and

332

after (120 min). Papp value of Lucifer Yellow was 6.4 × 10-7 cm/s and was in the range of the

333

values reported previously.30,40,41

334

Samples collected from transport wells were analyzed by UHPLC and confirmed the identity of

335

1 eluting at 4.8 min similar to that of 1 standard (Figure 3A). Peak identity was also verified by

336

using MS analysis, where a peak at m/z 321.4 was found in the basolateral solution (Figure S2A).

337

When the product ion at m/z 321.6 was fragmented, typical compound 1 product ions at m/z

338

302.6 and 285.3 were found and corresponded to the loss of one and two water molecules,

339

respectively (Figure S2B). These results allowed for further determination of 1 transport. As

340

shown in Figure 3B, 17.81 ± 0.66 µg of 1 were found in basolateral chamber (influx) after 120



Page 16 of 36

341

min, corresponding to 1.78 ± 0.06% of transport. A linear transport of 1 was found within tested

342

120 min. The apparent permeability coefficient from apical to basolateral direction influx (Papp a-

343

b)

344

b-a)

345

greater compared to the efflux rate (Papp b-a), suggesting that influx was the major transportation

346

route. Influx (Papp a-b) of compound 1 was greater in comparison to glucosamine (0.9 ± 0.5 × 10-6

347

cm/s).42 These results show that 1 can pass the transepithelial barrier and thus presumably have a

348

physiological function. Walle and Walle25 discovered that the carcinogenic heterocyclic amine

349

PhIP (Figure S1A) produced in the cooking process of various meat can also be efficiently

350

transported across the Caco-2 cell mono layer with Papp a-b of 15.1 ± 0.6 × 10-6 cm/s, while Chen

351

et al.43 showed that the anti-inflammatory heterocyclic compound F3-A (Figure S1B), originally

352

isolated from hexose-lysine Maillard reaction model and later from bread,26 can efficiently pass

353

across the Caco-2 cell monolayers (Papp a-b = 60.0 ± 0.35 × 10-6 cm/s). Even though the Papp of 1

354

is less than in previous studies25,26 taken together these studies indicate that the heterocyclic

355

compounds produced from nonenzymatic browning reactions can be absorbed by the intestinal

356

epithelial cells and exert their specific effects.

357

It is also of interest to compare the Papp

358

hydropic compounds. For instance, propranolol, a high permeability–high solubility lipophilic

359

drug, mainly absorbed by passive transcellular route with >90% bioavailability have a Papp a-b of

360

11.2 ± 0.5 × 10-6 cm/s.34 In contrast, furosemide (Papp = 2.20 ± 0.01 × 10-6 cm/s) is a low

361

permeability-high

362

bioavailability.34 Atenolol is another low permeability marker (Papp a-b = 0.50 ± 0.08 × 10-6 cm/s),

363

which is used in Caco-2 cell to compare the transport rate of an experimental compound.40 The

for 1 transport was 1.30 ± 0.02 × 10-6 cm/s and from basolateral to apical direction efflux (Papp was 0.60 ± 0.02 × 10-6 cm/s. These results indicate that the influx rate (Papp a-b) was 2.2 times

a-b

solubility hydrophilic

of 1 with the Papp

a-b

compound26 which

of known hydrophilic and

showed

10

-

60%

of


Page 17 of 36


364

transport rate of 1 (1.30 ± 0.02 × 10-6 cm/s) was closer to the value reported for furosemide and

365

2.8-fold greater compared to atenolol. There are several reports which indicate the correlation

366

between Papp a-b and extent of drug absorption in human. The most accepted theoretical based on

367

the correlation between in vitro obtained Papp

368

reported44 that compounds with permeability coefficients from 1 × 10-7 to 4 × 10-5 cm/s, have a

369

good correlation between the permeability coefficients in Caco-2 cells and percent absorbed in

370

humans. Being consistent with these studies, 1 may also have a good permeability and

371

bioavailability in in vivo conditions. However, this hypothesis needs to be tested under in vivo

372

conditions.

373

Mechanism of Fructosazine’s Transepithelial Transport in the Caco-2 Cell Line

374

Validation and rationale of the inhibitors used. How 1 is transported across the transepithelial

375

barrier is a question that we tried to address by using different inhibitors that specifically block

376

one process at a time. As reported in the introduction, we hypothesize that hexose transporters

377

are involved in the transport of 1 across the Caco-2 monolayer. There are two main types of

378

hexose transporters present in intestinal Caco-2 cell model, SGLT and GLUT, designated as

379

sodium-dependent glucose cotransporters and facilitative glucose transporters, respectively.

380

SGLT1 and 2 are very important cotransporters present on the apical surface of Caco-2 cells and

381

transport glucose molecule in exchange of sodium ions.45 To test this hypothesis, phlorizin was

382

used as a potential inhibitor of SGLT1 and 2 transporters, since it specifically inhibits both

383

SGLT1 and 246 without effecting the GLUT family transporters.47,48 Phloretin and quercetin have

384

been used to inhibit GLUT1 and GLUT2 transporters.49-51 Wortmannin and cytochalasin D have

385

also been used to understand if transcytosis or tight junction processes are involved in compound

386

1 transport.

a-b

and in vivo drug absorption in human had



Page 18 of 36

387

Though these inhibitors are widely used and tested in Caco-2 cell models, a validation was

388

performed to verify the efficiency of inhibitors in the presence of radioactive [14C]glucose

389

(Figure S3A). A linear transport of [14C]glucose was demonstrated up to 2 h and the Papp a-b of

390

[14C]glucose was 49.8 ± 1.2 × 10-6 cm/s. This value was close to the one reported by Walgren et

391

al.39 who obtained Papp

392

performance of the Caco-2 cell system in context of existing literature. As shown in Figure S3A

393

addition of GLUT (phloretin, quercetin) and SGLT (phlorizin) inhibitors can decrease glucose

394

transport up to 98.8, 98.6 and 68.7% respectively. On the contrary, the addition of a transcytosis

395

inhibitor (wortmannin) did not interfere with glucose transport. The presence of a tight junction

396

disruptor, cytochalasin D, resulted in an increased transport rate up to 4.16%. Modified cell lines,

397

–GLUT decreased 99.4% of glucose transport, while the overexpression of GLUT5 (++GLUT5)

398

did not affect the rate of [14C]glucose transport (Figure S3A). The cytotoxic effect of these

399

inhibitors on the Caco-2 cell model was also considered. Here, the results are shown in Figure

400

S2B, where no cytotoxicity was found, indicating that the applied concentration can be used

401

without affecting cell integrity.

402

GLUT and SLGT1 Families in Compound 1 Transport. The cumulative transport of 1 after 120

403

min without any inhibitor (control) was 17.8 ± 0.7 µg (Figure 3). No reduction in cumulative

404

transport as compared to control was found by using transcytosis inhibitor wortmannin and was

405

18.41 ± 0.42 µg after 120 min. Even though a cytochalasin D treatment increased the transport of

406

1 to 20.01 ± 0.77 µg corresponding to about a 12% increment, no difference (p > 0.05) was

407

found as compared to the control (Figure 3C). These results indicate no involvement of a gap

408

junction-mediated transport in transport of 1. A significant decrease of 45.2% was found in 1

409

cumulative transport when phlorizin was used (0.97 ± 0.07%) after 120 min as compared to the

a-b

of 36.8 ± 1.1 × 10-6 cm/s for glucose transport. This validates the


Page 19 of 36


410

control. This suggests an important role of SGLT family of transporters in 1 transport. The

411

addition of phloretin and quercetin (GLUT1 and 2 inhibitor) significantly decreased the transport

412

rate of 1 to 5.65 ± 0.15 µg (0.56 ± 0.01%) and 3.95 ± 0.33 µg (0.39 ± 0.03%), respectively

413

(Figure 3C), representing a reduction of 68.3 and 77.8%, respectively. Hence, the experimental

414

data indicates that 1 is mainly transported through GLUT and SGLT1 across the Caco-2 cells.

415

To further confirm the involvement of the hexose transporter families and to understand

416

which among them is responsible for the transport of 1, two new Caco-2 cell lines were

417

produced. In the first cell line, the down regulation of GLUT and SGLT1 (-GLUT cell line)

418

families was achieved, while in the second cell line the overexpression of GLUT5 (++GLUT5

419

cell line) was accomplished according to Mesonero et al.38 Western blotting and image analysis

420

were used to evaluate the expression of GLUT1, GLUT2, GLUT 3, GLUT4 and GLUT5 and

421

SLGT1 in these two new cell lines and the results are reported in Figure 4. Protein expression

422

data was in agreement with Miguel et al.32 and suggested a down regulation of all GLUT (1-5)

423

and SGLT transporters in –GLUT cell line (Figure 4A, B). Fructose supplemented cell line,

424

++GLUT5, showed a two-fold increase in GLUT5 expression when compared to control cells

425

while expression of other GLUT and SGLT1 transporters were not significantly changed (Figure

426

S4 A,B). Transportation studies were further performed using these cell lines (Figure 5A).

427

Experimental data suggested that –GLUT cells line was unable to transport 1 after 120 min,

428

while ++ GLUT5 cell line transported 18.46 ± 1.40 µg of compound 1 after 120 min, which was

429

not different from the control (Figure 5A). This indicates that hexose transporter family is

430

involved in 1 transport with the main role of GLUT1 or GLUT2 and SGLT. Radioactive

431

[14C]glucose (3.4 µM) in presence or absence of 1 (2 mg/mL) was also used to study the

432

competition between these two molecules in term of transport rate. As reported in earlier section,



Page 20 of 36

433

[14C]glucose transport rate was 49.8 ± 1.2 × 10-6 cm/s in the absence of 1 (Figure 5B), while

434

presence of 1 significantly (p < 0.05) reduced the glucose transport rate (24.2 ± 0.9 × 10-6 cm/s).

435

This suggests that 1 directly competes with glucose transport, which indirectly suggests these

436

two molecules may share common transport route.

437

The transport of glucose, especially via GLUT1, has been studied exhaustively. It has

438

been proposed that the hydroxyl- and amide-containing amino acid side chains within

439

transmembrane helices of GLUT1 form the sugar binding sites via hydrogen bond formation

440

with glucose hydroxyl groups.27 Interestingly, glucose derivatives in which a hydroxyl group

441

configuration was inverted or replaced at C-1, C-2, C-3, C-4 or C-6 of the D-glucose, were all

442

bound to the carrier, indicating that no specific hydroxyl group was responsible for the binding

443

event.29 Hydrophobic interactions between aromatic amino acid side chains and the C-6 region of

444

glucose were also emphasized.28 By comparing the transport of deoxy sugars with their

445

fluorinated derivatives, C-l-, C-3-, and C-6-hydroxyls of glucose were found to be involved in

446

binding to the transporter.28 According to docking studies conducted by Salas-Burgos et al.52

447

glucose rolls through the GLUT1 channel by forming approximately one H-bond at a time,

448

migrating along the channel by rolling along the wall, forming a new H-bond forward as the one

449

in the back is being broken. The formation of more than one/two H-bond at a time can stabilize

450

the substrate in position, keeping it in place rather than facilitating migration. These authors also

451

emphasized the role of the hydrophobic interactions between the pyranose ring of glucose and

452

the aromatic amino acids present in the GLUT1 channel. Therefore both hydrophilic and

453

hydrophobic interactions are important for the migration of glucose along the GLUT1 channel.

454

Compound 1 possess both hydrophilic (four –OH groups) and hydrophobic (pyrazine) residues


Page 21 of 36


455

making it a possible candidate to roll through GLUT transporters with both hydrophilic and

456

hydrophobic interactions.

457

Anthocyanins, which occur primarily as glycosides of their respective aglycone

458

anthocyanidin chromophores with the sugar moiety attached at the position 3 on the C ring or at

459

the 5- or 7- position on the A ring,53 were also shown to be transported through hexose

460

transporters.53,54 For instance, anthocyanins from red grape skin were transported via GLUT2,

461

while neither GLUT5 nor SGLT1 were involved.53 Yi et al.55 investigated the effects of different

462

aglycones, sugar moieties and chemical structures of different types of anthocyanins and found

463

that blueberries anthocyanins are transported through the Caco-2 cell monolayers with relatively

464

less efficiency compared to other aglycone polyphenols. Although it was originally believed that

465

anthocyanins needed to be hydrolyzed to an aglycone form before they can be absorbed,53 the

466

results from Miyazawa et al.56 indicated that in mammals anthocyanins are incorporated in intact

467

glycoside forms, from the digestive tract into the blood circulation system. These studies indicate

468

that hexose transporter may have the potential to transport more complex and larger MW

469

compounds than hexose.

470

In the current study 1 can be transported across the Caco-2 cells monolayers, and the preliminary

471

inhibitory studies indicated the involvement of hexose transporters (GLUT and SGLT).

472

Compound 1 is produced from autocondensation reaction of glucosamine. The presence of both

473

hydroxyl groups and hydrophobic pyrazine ring makes 1 a good candidate for hexose transport

474

as similar to glucose. Further studies are needed to understand the mode of recognition.

475

Supporting Information

476

Figure S1. Chemical structures of (A) 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)

477

and (B) 5-(5,6-dihydro-4H-pyridin-3-ylidenemethyl)furan-2-yl]-methanol (F3-A); Figure S2.



Page 22 of 36

478

Mass spectrometric characterization of the basolateral solution in Caco-2 cell monolayers; Figure

479

S3. Cumulative amount transported (ng) of [14C]glucose across Caco-2 cell monolayer in

480

presence or absence of different inhibitors (A) and cell viability in presence of different

481

inhibitors (MTT assay) (B); Figure S4. Western blot analysis of GLUT and SGLT hexose

482

transporters in (A) ++GLUT5 cell line and (B) their relative band intensity quantification; Table

483

S1. TEER values of the Caco-2 cell monolayers before and after transport experiments.

484

Funding Sources

485

This research was funded by grant from Natural Sciences and Engineering Research Council of

486

Canada (NSERC).

487

Abbreviations

488

DMEM, Dulbecco’s modified Eagle’s medium; EDTA, ethylenediaminetetraacetic acid; GLUT,

489

glucose transporters; HEPES, 4-(2 hydroxyethyl)-1-piperazineethanesulfonic acid; FBS, Fetal

490

bovine serum; HBSS, Hank’s balanced salt solution; HPLC, high performance liquid

491

chromatography; SGLT, Sodium-dependent glucose cotransporter; TEER, transepithelial

492

resistance;

493 494 495 496 497 498 499 500


Page 23 of 36


501

References

502 503 504 505 506

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[45] Mackenzie, B.; Loo, D. D. R., Panayotova-Heiermann, M.; Wright, E. M. Biophysical characteristics of the pig kidney Na+-glucose cotransporter SGLT2 reveal a common mechanism for SGLT1 and SGLT2. J. Biol. Chem. 1996, 271, 32678–32683.

631 632 633

[46] Panayotova-Heiermann, M.; Loo, D. D. R.; Wright, E. M. Kinetics of steady-state currents and charge movements associated with the rat Na+/glucose cotransporter. J. Biol. Chem. 1995; 270, 27099–27105.

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[47] Bell, G.I.; Kayanom, T.; Busem, J. Molecular biology of mammalian glucose transporters. Diabetes Care. 1990, 13, 198–208.

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[48] Ehrenkranz, J.R.; Lewis, N.G.; Kahn, C.R.; Roth, J. Phlorizin: a review. Diabetes Metab. Res. Rev. 2005, 21, 31-38.

638 639 640

[49] Yonemochi, H.I.; Nakatomi, M., Harada, H.; Takata, H.; Baba, O.; Ohshima, H.; Glucose uptake mediated by glucose transporter 1 is essential for early tooth morphogenesis and size determination of murine molars. Dev. Biol. 2012, 363, 52–61.

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[50] Walker, J.; Jijon, H.B.; Diaz, H.; Salehi, P.; Churchill, T.; Madsen, K.L.5-Aminoimidazole4-carboxamide riboside (AICAR) enhances GLUT2-dependent jejunal glucose transport: a possible role for AMPK. Biochem. J. 2005, 385, 485–491.

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[51] Song, J.; Kwon, O.; Chen, S.; Daruwala, R.; Eck, P.; Park, J.B., Levine. M. Flavonoid inhibition of sodium-dependent vitamin C transporter 1 (SVCT1) and glucose transporter isoform 2(GLUT2), intestinal transporters for vitamin C and glucose. J. Biol. Chem. 2002, 277, 15252-60.

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[52] Salas-Burgos, A.; Iserovich, P.; Zuniga, F.; Vera, J. S.; Fischbarg, J. Predicting the threedimensional structure of the human facilitative glucose transporter Glut1 by a novel evolutionary homology strategy: insights on the molecular mechanism of substrate migration, and binding sites for glucose and inhibitory molecules. Biophys. J. 2004, 87, 2990–2999.

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[53] Faria, A.; Pestana, D.; Azevedo, J.; Martel, F.; de Freitas, V.; Azevedo, I.; Mateus, N.; Calhau, C. Absorption of anthocyanins through intestinal epithelial cells – putative involvement of GLUT2. Mol. Nutr. Food Res. 2009, 53, 1430-1437.

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[54] Kamiloglu, S.; Capanoglu, E.; Grootaert, C.; Camp, J. V. Anthocyanin absorption and metabolism by human intestinal Caco-2 cells-a review. Int. J. Mol. Sci. 2015, 16, 21555– 21574.



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[55] Yi, W.; Akoh, C. C.; Fischer, J.; Krewer, G. Absorption of anthocyanins from blueberry extracts by caco-2 human intestinal cell monolayers. J. Agric. Food Chem. 2006, 54, 56515658.

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[56] Miyazawa, T.; Nakagawa, K.; Kudo, M.; Muraishi, K.; Someya, K. Direct intestinal absorption of red fruit anthocyanins, cyanidin-3-glucoside and cyanidin-3,5-diglucoside, into rats and humans. J. Agric. Food Chem. 1999, 47, 1083-1091.

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683


Figure Captions

684 685

Figure 1. The central role of aminocarbonyls in the production of pyrazines. General mechanism

686

of pyrazine formation from aminocarbonyl derived from (A) Strecker degradation (partly

687

adapted from Scalone et al.3) or (B) from fructose-ammonia reaction system.

688 689

Figure 2. Effect of fructosazine on Caco-2 cells viability as determined by MTT cell proliferation

690

assay. Data are presented as mean value ± SD (n = 3). The mean cell viability followed by

691

different letters indicates significant differences (p ≤ 0.05).

692 693

Figure 3. (A) Representative analytical UHPLC-UV chromatograms of 1 collected from

694

basolateral chamber from 0 to 120 min. (B) Cumulative quantity (µg) of 1 transported from

695

(circle) the apical (donor) to basolateral (acceptor) or (triangle) basolateral-to-apical sides of

696

Caco-2 cell monolayers up to 120 min. (C) Effects of different transporter inhibitors on apical-

697

to-basolateral transport of 1 (2 mg/mL). The data are presented as mean ± SD (n = 3). The mean

698

basolateral transport of 1 in the presence or absence of inhibitors followed by different letters

699

indicates significant differences (p ≤ 0.05).

700 701

Figure 4. Western blot analysis of GLUT and SGLT hexose transporters in (A) –GLUT cell line

702

and (B) their relative band intensity quantification presented as β-actin ratio. The data are

703

represented as mean value ± SD (n = 3).

704 705



Page 30 of 36

706

Figure 5. A) Cumulative quantity transported (µg) of 1 across Caco-2 cell monolayer up to 120

707

min in control and modified (–GLUT, ++GLUT5) cell lines. B) Cumulative transport (ng) of

708

radioactive [14C]glucose in absence (circle) or presence (square) of 1 (2 mg/mL) across Caco-2

709

cell monolayer. The data are presented as mean value ± SD (n = 3). ND refers to not detected.

710 711 712 713 714 715 716 717 718 719 720 721 722 723


Page 31 of 36


Figure 1.

A :B

-2H O

-2H

2

Path I Pyrazine

Dihydropyrazine α-aminocarbonyls from Strecker degradation

Base attack (:B) Path II -

R 5

-H O

H+

2

R =H 4

B:

Pyrazine

B:

B -H O

NH

2

3

Glucosamine (α-aminocarbonyl)

-2H O

-HO

2

2

Dihydropyrazine - 2H

Fructosazine (compound 1)

Deoxyfructosazine



Page 32 of 36

Figure 2. a

c d

e

f

0 0. 5 1 1. 5 2 2. 5 3 3. 6 4 4. 6 5. 6 7. 5 10

Cell viability (%)

b


Page 33 of 36


Figure 3. A

Absorbance

120 min 90 min 60 min 30 min 0 min Standard

min

B

C a

a

b c



Page 34 of 36

Figure 4. A

β-actin

GLUT1

GLUT2

GLUT3

GLUT4

GLUT5

SGLT1

B

0.4

SG LT 1

T5 G LU

T4 G LU

T3 G LU

T2 G LU

T1

0.0

G LU

Retalive band intensity of transporters/β-actin

0.8


Page 35 of 36


Figure 5. B

A

ND



Page 36 of 36

Categories: First choice: Bioactive Constituents and Functions; Second choice: Food and Beverage Chemistry/Biochemistry; Third choice: Chemical Aspects of Biotechnology/Molecular Biology


Transport of the Glucosamine-Derived Browning Product Fructosazine

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